Proteases are a large group of proteins that comprise approximately 2% of all gene products (Rawlings and Barrett, 1999). Proteases catalyse the hydrolysis of peptide bonds and are vital for the proper functioning of all cells and organisms. Proteolytic processing events are important in a wide range of cellular processes including bone formation, wound healing, angiogenesis and apoptosis.
The lysosomal cysteine proteases were initially thought to be enzymes that were responsible for non-selective degradation of proteins in the lysosomes. They are now known to be accountable for a number of important cellular processes, having roles in apoptosis, antigen presentation, coagulation, digestion, pro-hormone processing and extracellular matrix remodelling (Chapman et al, 1997).
Cathepsin S (Cat S) is a member of the papain superfamily of lysosomal cysteine proteases. To date, eleven human cathepsins have been identified, but the specific in vivo roles of each are still to be determined (Katunuma et al, 2003). Cathepsins B, L, H, F, O, X and C are expressed in most cells, suggesting a possible role in regulating protein turnover, whereas Cathepsins S, K, W and V are restricted to particular cells and tissues, indicating that they may have more specific roles (Kos et al, 2001; Berdowska, 2004, Clinica Chimica Acta. 2004; 342: 41-69).
Cat S was originally identified from bovine lymph nodes and spleen and the human form cloned from a human macrophage cDNA library (Shi et al, J Biol. Chem. 1992; 267: 7258-7262). The gene encoding Cat S is located on human chromosome 1q21. The 996 base pair transcript encoded by the Cat S gene is initially translated into an unprocessed precursor protein with a molecular weight of 37.5 kDa. The unprocessed protein is composed of 331 amino acids; a 15 amino acid signal peptide, a 99 amino acid pro-peptide sequence and a 217 amino acid peptide. Cat S is initially expressed with a signal peptide that is removed after it enters the lumen of the endoplasmic reticulum. The propeptide sequence binds to the active site of the protease, rendering it inactive until it has been transported to the acidic endosomal compartments, after which the propeptide sequence is removed and the protease is activated (Baker et al, 2003 Protein Expr Purif. 28, 93-101).
Cat S has been identified as a key enzyme in major histocompatibility complex class II (MHC-II) mediated antigen presentation, by cleavage of the invariant chain, prior to antigen loading. Studies have shown that mice deficient in Cat S have an impaired ability to present exogenous proteins by APCs (Nakagawa et al, Immunity. 1999; 10: 207-217). The specificity of Cat S in the processing of the invariant chain Ii, allows for Cat S specific therapeutic targets in the treatment of conditions such as asthma and autoimmune disorders (Chapman et al, 1997).
Pathological Association of Cat S
Alterations in protease control frequently underlie many human pathological processes. The deregulated expression and activity of the lysosomal cysteine protease Cathepsin S has been linked to a range of conditions including neurodegenerative disorders, autoimmune diseases and certain malignancies.
Cat S upregulation has been linked to several neurodegenerative disorders. It is believed to have a role in the production of the β peptide (Aβ) from the amyloid precursor protein (APP) (Munger et al, Biochem. J. 1995; 311: 299-305) and its expression has been shown to be upregulated in both Alzheimer's Disease and Down's Syndrome (Lemere et al, 1995). Cat S may also have a role in Multiple Sclerosis through the ability of Cat S to degrade myelin basic protein, a potential autoantigen implicated in the pathogenesis of MS (Beck et al, 2001, Eur. J. Immunol. 2001; 31: 3726-3736) and in Creutzfeldt-Jakob disease (CJD) patients, Cat S expression has been shown to increase more than four fold (Baker et al, 2002).
Cathepsin S has been reported to be overexpressed in atherosclerotic and restenosis after angioplasty (Cheng et al, Am. J Pathology, 2006, 168: 685-694). In these conditions, the CatS was reported to co-localise with integrin αvβ3 as a receptor on the vascular smooth muscle cell surface.
Angiogenesis, the development of microvasculature, is an integral process within many normal physiological processes such as normal development and wound healing. Angiogenesis is characterised by the stimulation of endothelial cells to form primary blood vessels where a non-clarified complex interplay exists between the endothelial cells, surrounding microenvironment and a range of pro- and anti-angiogenic factors. However, uncontrolled or inappropriate angiogenesis is accepted as an underlying factor to the pathology of a wide range of diseases including tumour progression and ocular disease.
The association of CatS with angiogenesis was first shown in vitro using CatS deficient endothelial cells (Shi et al, 2003). Microvascular endothelial cells (ECs) have been shown to secrete proteases, permitting penetration of the vascular basement membrane as well as the interstitial extracellular matrix. Treatment of cultured ECs with inflammatory cytokines or angiogenic factors stimulated expression of CatS, and its inhibition reduced microtubule formation. CatS−/− mice displayed defective microvessel development during wound repair in comparison to wild-type controls (Shi et al, 2003).
Further examination of the role of CatS in angiogenesis and tumour growth was demonstrated in a transgenic mouse model for pancreatic islet cell carcinoma. CatS−/− mice were found to develop significantly smaller tumours and fewer angiogenic islets in comparison to the CatS+/+ control mice (Gocheva et al., 2006). Insight to the molecular mechanism underpinning this phenotype was subsequently provided by evidence that CatS could cleave and inactivate anti-angiogenic peptides and promote the generation of active pro-angiogenic fragments (Wang et al, 2006).
The role of Cat S has also been investigated in specific malignancies. The expression of Cat S was shown to be significantly greater in lung tumour and prostate carcinomas sections in comparison to normal tissue (Kos et al, 2001, Fernandez et al, 2001) and suggested that Cat S may have a role in tumour invasion and disease progression.
Recent work on Cat S demonstrated the significance of its expression in human astrocytomas (Flannery et al, 2003). Immunohistochemical analysis showed the expression of Cat S in a panel of astrocytoma biopsy specimens from WHO grades I to IV, but appeared absent from normal astrocytes, neurones, oligodendrocytes and endothelial cells. Cat S expression appeared highest in grade IV tumours and levels of extracellular activity were greatest in cultures derived from grade IV tumours.
Cat S has been shown to be active in the degradation of ECM macromolecules such as laminin, collagens, elastin and chondroitin sulphate proteoglycans (Liuzzo et al, 1999). Using invasion assays with the U251MG grade IV glioblastoma cell line, a 61% reduction in invasion in the presence of a Cat S inhibitor LHVS29 has been shown (Flannery et al, 2003).
The generation of inhibitors specifically targeting Cat S have potential as therapeutic agents for alleviations of the symptoms associated with the activity of this protease.
The implication of aberrant extracellular cysteine cathepsin activity in tumour progression has been of particular focus to researchers. Each of these lysosomal enzymes has been implicated in the progression of various tumours, where it is thought that their abnormally high secretion from tumour cells leads to the degradation of the extracellular matrix (ECM). This aberrant breakdown of ECM components such as elastin and collagen accelerates the penetration and invasion of these abnormal cells to surrounding normal tissue. Additionally, roles in angiogenesis and the processing of other molecules have also been attributed to inappropriate cathepsin activity (Lah and Kos, 1998 Biol. Chem. 379, 125-30; Folkman and Ingber, 1992; Fernandez et al, 2001).
Much research has focussed on the underlying mechanisms that result in this devastating increase in extracellular proteolytic activity. Cathepsins are believed to be involved in the degradation of the ECM directly through their ability to degrade components of the ECM such as laminin, fibronectin and collagen or indirectly through the activation of other proteases in a proteolytic cascade (Koblinski et al, 2000; Rao et al, 2003).
Inhibition of Cat S
When proteases are over-expressed, therapeutic strategies have focused on the development of inhibitors to block the activity of these enzymes. The generation of specific small molecule inhibitors to the cathepsins have proved difficult in the past, due to problems with selectivity and specificity. The dipeptide α-keto-β-aldehydes developed as potent reversible inhibitors to Cat S by Walker et al, had the ability to inhibit Cat B and L, albeit with less efficiency (Walker et al, Biochem. Biophys. Res. Comm. 2000; 75: 401-405), and the Cat S inhibitor 4-Morpholineurea-Leu-HomoPhe-vinylsulphone (LHVS) has also been shown to inhibit other cathepsins when used at higher concentrations (Palmer et al, J. Med. Chem. 1995; 38: 3193-3196).
A broad range of antineoplastics have been developed for the treatment of cancer. However, although many of these compounds are successfully used in treatment strategies, in many cases a particular treatment regime does not result in complete clearance of neoplasms or only does so temporarily. There thus remains a great need for the development of new cancer treatments and therapeutic regimes.